The present study was designed to investigate the dietary effects of protease and prebiotic mixtures with free fishmeal diets on red tilapia (Oreochromis sp.). One hundred and fifty fish weighing 7.40±0.05g were randomly divided into four experimental treatments in 3 replicates. Four experimental diets were designed containing different inclusion levels of protease as 0%, 0.10%, 0.15%, and 0.20% named T1, T2, T3, and T4, respectively beside control with prebiotic mixtures was 2 g kg^-1. All fish were fed 3% of the body weight daily for 84 days. Liver and visceral weights were measured to determine relative gut length, hepatosomatic index (HSI) and viscerosomatic index (VSI) indices. HSI and VSIwere higher in the groups fed with protease with free fishmeal diets compared to the control group especially in T4 and T3, respectively. The results showed a significant increase in ALT, AST, ALP, TG and TP in T3 and T4 groups compared to the control group. In addition, significant enhancement was observed in T4 of villi numbers, goblet cell numbers and villi width. Furthermore, the control group showed the highest villi length but the higher muscular layer thickness was recorded for T2 group. By intestinal investigation, the results show an improvement of T2, T3 and T4 groups but T1 group has villi shortening and broadening. The results of liver histology show an improvement of T3 and T4 and mild improvement with moderate inflammation in some tissues in T1 and T2 groups. The results of spleen histology show an improvement of T2, T3 and T4 groups with some congestion in some tissues but T1 group has a hemorrhage and some congestion in some tissues. In conclusion, the increasing protease level with prebiotic mixtures can effectively improve the physiological status and health of red tilapia (Oreochromis sp.).